Steven R. Lear

ACAT-2, A Second Mammalian Acyl-CoA:Cholesterol Acyltransferase ITS CLONING, EXPRESSION, AND CHARACTERIZATION

The synthesis of cholesterol esters by acyl-CoA:cholesterol acyltransferase (ACAT, EC 2.3.1.26) is an important component of cellular cholesterol homeostasis. Cholesterol ester formation also is hypothesized to be important in several physiologic processes, including intestinal cholesterol absorption, hepatic lipoprotein production, and macrophage foam cell formation in atherosclerotic lesions. Mouse tissue expression studies and the disruption of the mouse ACAT gene (Acact) have indicated that more than one ACAT exists in mammals and specifically that another enzyme is important in mouse liver and intestine. We now describe a second mammalian ACAT enzyme, designated ACAT-2, that is 44% identical to the first cloned mouse ACAT (henceforth designated ACAT-1). Infection of H5 insect cells with an ACAT-2 recombinant baculovirus resulted in expression of a ∼46-kDa protein in cell membranes that was associated with high levels of cholesterol esterification activity. Both ACAT-1 and ACAT-2 also catalyzed the esterification of the 3β-hydroxyl group of a variety of oxysterols. Cholesterol esterification activities for ACAT-1 and ACAT-2 exhibited different IC50 values when assayed in the presence of several ACAT-specific inhibitors, demonstrating that ACAT inhibitors can selectively target specific forms of ACAT. ACAT-2 was expressed primarily in mouse liver and small intestine, supporting the hypothesis that ACAT-2 contributes to cholesterol esterification in these tissues. The mouse ACAT-2 gene (Acact2) maps to chromosome 15 in a region containing a quantitative trait locus influencing plasma cholesterol levels. The identification and cloning of ACAT-2 will facilitate molecular approaches to understanding the role of ACAT enzymes in mammalian biology.

The Effect of Diabetes Mellitus on Sterol Synthesis in the Intact Rat

Diabetes mellitus in both humans and animals is characterized by elevations in plasma cholesterol concentrations. The cause of this hypercholesterolemia is unknown. The present study employed tritiated water to quantity sterol synthesis in intact diabetic and control animals. Sterologenesis was 60–246% greater in the gut of diabetic animals than in controls. This enhancement of sterol synthesis occurred soon after the onset of diabetes and persisted for at least 3 wk. Moreover, insulin therapy markedly decreased gut sterol synthesis in diabetic animals to levels only slightly greater than in controls. Diabetes increased sterol synthesis primarily in the small intestine, but a small increase was also observed in the large intestine. Subfractionation of the small intestine into epithelial cell and muscularis cell layers revealed an increased sterologenesis in both layers. Quantitatively, though, the epithelial layer accounted for the majority of the enhancement of small intestine sterol synthesis observed in diabetic animals. De novo sterologenesis in tissues other than the intestines (liver, skin, stomach, or remaining carcass) was not significantly altered by diabetes. This study demonstrates that diabetes markedly stimulates sterol synthesis, an effect that is specifically localized to the intestines.

De novo sterologenesis in the intact rat

On the basis mainly of in vitro studies, the liver and, to a lesser degree, the small intestine are widely accepted as the major sites of de novo sterologenesis. Utilizing [3H]water, we have investigated de novo sterologenesis in intact rats. Greater than 80% of labeled nonsaponifiable lipids and more than 70% of the labeled cholesterol were localized to extrahepatic, extraintestinal tissues. Feeding cholesterol markedly suppressed hepatic sterologenesis but had little influence on extrahepatic sites of sterol synthesis. Similarly, partial hepatectomy, which greatly decreased sterol synthesis in the liver, also did not significantly affect the accumulation of labeled sterols in extrahepatic tissues; therefore, the transport of sterols from the liver did not account for a significant portion of labeled sterols in extrahepatic tissues. Cannulation of the thoracic duct demonstrated that transport of newly synthesized intestinal sterols to peripheral tissues also did not account for the large accumulation of labeled sterols in extrahepatic, extraintestinal tissues. The primary extrahepatic, extraintestinal sites of sterologenesis were the skin and remaining carcass. The lung, kidney, spleen, heart, ovary, brain, muscle and adipose tissue made minor contributions to de novo sterol synthesis. Therefore, tissues other than the liver and intestine, especially the skin and remaining carcass, are important sites of de novo sterologenesis in vivo.

Profiling sterols in cerebrotendinous xanthomatosis: Utility of Girard derivatization and high resolution exact mass LC―ESI-MSn analysis

In this study we profile free 3-oxo sterols present in plasma from patients affected with the neurodegenerative disorder of sterol and bile acid metabolism cerebrotendinous xanthomatosis (CTX), utilizing a combination of charge-tagging and LC-ESI-MS(n) performed with an LTQ-Orbitrap Discovery instrument. In addition, we profile sterols in plasma from 24-month-old cyp27A1 gene knockout mice lacking the enzyme defective in CTX. Charge-tagging was accomplished by reaction with cationic Girards P (GP) reagent 1-(carboxymethyl) pyridinium chloride hydrazide, an approach uniquely suited to studying the 3-oxo sterols that accumulate in CTX, as Girards reagent reacts with the sterol oxo moiety to form charged hydrazone derivatives. The ability to selectively generate GP-tagged 3-oxo-4-ene and 3-oxo-5(H) saturated plasma sterols enabled ESI-MS(n) analysis of these sterols in the presence of a large excess (3 orders of magnitude) of cholesterol. Often cholesterol detected in biological samples makes it challenging to quantify minor sterols, with cholesterol frequently removed prior to analysis. We derivatized plasma (10 μl) without SPE removal of cholesterol to ensure detection of all sterols present in plasma. We were able to measure 4-cholesten-3-one in plasma from untreated CTX patients (1207±302 ng/ml, mean±SD, n=4), as well as other intermediates in a proposed pathway to 5α-cholestanol. In addition, a number of bile acid precursors were identified in plasma using this technique. GP-tagged sterols were identified utilizing high resolution exact mass spectra (±5 ppm), as well as MS(2) ([M](+)→) spectra that possessed characteristic neutral loss of 79Da (pyridine) fragment ions, and MS(3) ([M](+)→[M-79](+)→) spectra that provided additional structurally informative fragment ions.

De novo cholesterol synthesis in three different animal models of diabetes

SummaryRecent studies have demonstrated that cholesterol synthesis is increased two to threefold in the intestines of streptozotocin-induced diabetic rats. Cholesterol synthesis in tissues other than the intestines, including the liver, was not significantly altered by diabetes. In diabetic Chinese hamsters, cholesterol synthesis was increased 2.5-fold in both the small and large intestine. These observations are similar to our findings in diabetic rats and suggest that a stimulation of intestinal cholesterogenesis may be a uniform phenomenon in insulinopenic diabetes. In db/db mice, cholesterol synthesis was increased in both the liver and intestines but quantitatively the increase in hepatic cholesterogenesis was of much greater magnitude. Cholesterol feeding, which markedly inhibited hepatic cholesterol synthesis in both control and db/db mice, did not obliterate this difference in hepatic cholesterol synthesis. In ob/ob mice, the severity of the metabolic disturbances was less than that observed in db/db mice and no abnormalities in cholesterol synthesis were observed in animals ingesting a low cholesterol diet. However, in ob/ob mice fed a high cholesterol diet, hepatic cholesterol synthesis was increased. These observations suggest that in obese insulin resistant diabetic animals of milder severity, the abnormality in hepatic cholesterol synthesis manifests itself only when the animals are ingesting a high cholesterol diet. The results of this and previous studies suggest that in insulinopenic diabetes there is a stimulation of cholesterol synthesis that is localized to the intestines, whereas in obese, insulin-resistant diabetic animals, cholesterol synthesis is altered in the liver.

The effect of diabetes mellitus on the lymphatic transport of intestinal sterols

Previous studies have demonstrated that in intact animals, de novo cholesterol synthesis is increased in the small and large intestines of streptozotocin-induced diabetic rats. The aim of this study was to determine if this enhanced intestinal cholesterol synthesis in diabetic animals results in the increased transport of newly synthesized sterols from the intestines to the peripheral circulation. Thoracic ducts were cannulated in control and diabetic animals and the quantity of newly labeled sterols in the lymph determined after the administration of tritiated water. Labeled sterols in the 24-hour lymph drainage were increased fourfold in the diabetic animals as compared to control animals. Additionally, the percentage of newly synthesized sterols synthesized in the small intestine and transported by the lymph is almost twofold greater in the diabetics. Thus, the increased quantity of labeled sterols present in the thoracic duct lymph of diabetic animals is accounted for by two factors, an enhancement of small intestinal sterol synthesis in diabetic animals and the increased percentage transport of newly synthesized sterols from the small intestine to the bloodstream. In both control and diabetic animals, chylomicrons were the major lipoprotein fraction in which the newly synthesized sterols were transported in the lymph. These findings demonstrate that the increased sterologenesis observed in the intestine of diabetic animals results in the increased transport of newly synthesized cholesterol from the intestines to the circulation.

The Effect of Cholesterol Feeding and Alterations in Bile Acid Homeostasis on De Novo Sterologenesis in Diabetic Rats

Previous studies have demonstrated that de novo cholesterol synthesis is increased two- to threefold in the intestines of diabetic animals. This increase is due to a stimulation of cholesterogenesis in both the small and large intestine but, quantitatively, the small intestine is primarily responsible for the observed increase. The present study examined the effect of cholesterol feeding and alterations of bile acid homeostasis on de novo sterol synthesis in intact normal and diabetic animals. Cholesterol feeding in the control animals did not affect sterol synthesis in the small intestine, but in diabetic animals cholesterol feeding markedly inhibited small intestinal sterologenesis. The threefold stimulation of small intestinal sterol synthesis observed in diabetic animals is completely obliterated by cholesterol ingestion. Moreover, this inhibition of sterol synthesis by cholesterol feeding in the small intestine of diabetic animals occurred very rapidly (within 36 h). In the large intestine, cholesterol feeding did not influence sterol synthesis in either the diabetic or control animals. In the liver, cholesterol feeding markedly inhibited sterol synthesis to similar degrees in the diabetics and controls. Colestipol feeding and biliary drainage, procedures that reduce bile acid pool size, stimulated sterol synthesis in the liver and small intestine of both diabetic and control animals. However, reductions in bile acid pool size increased sterologenesis in the large intestine in control animals but had no effect in the diabetics. Bile acid ingestion did not alter either small or large intestinal sterologenesis in the diabetic or control animals. In conclusion, the present study demonstrates the sterol synthesis is enhanced in the small and large intestine of diabetic animals and, moreover, both the cholesterol- and bile acid-mediated regulation of cholesterol synthesis in the intestines of the diabetic animals is altered from normal.